There is a fundamental lack of information regarding the chemical mechanisms by which glycosyl transfer and the incorporation of glycosylation patterns into the final target (proteins, cell walls and/or antibiotics) occur due primarily to the restricted availability of glycosyltransferases and their nucleotide diphosphate sugar substrates. While essentially all research in this arena has focused upon eukaryotic transferases, we suggest prokaryotic glycosyltransferases to be a rich alternative source with perhaps better accessibility. Thus we propose a detailed comparison of two prokaryotic model mannosyltransferases which use identical substrates but differ only in the stereochemistry of mannosyltransfer (alpha1 yields 4 or "retaining" versus beta1 yields 4 or "inverting"; Man Talpha4 and Man Tbeta4, respectively). Through a multi-disciplinary approach (overexpression and purification of Man Talpha4 and Man Tbeta4; simplified assay systems based upon synthetic "unnatural" acceptors; chemical mechanistic probes including substrate and solvent isotope effects, elucidation/characterization of covalent Man T alpha4- and/or Man Tbeta4- substrate complexes, nucleophilic traps and testing the catalytic competency of synthetic substrate analogs; and Man Talpha4/Man Tbeta4 structural probes including site-directed mutagenesis; in vitro molecular evolution; differential labeling and/or active site-directed affinity labeling), the proposed work should help define i) the Man Talpha4 and Man Tbeta4 chemical reaction mechanisms and ii) how the Man Talpha4 and Man Tbeta4 scaffold governs these important reactions. Using mannosyl transfer as the model, the proposed work will serve to establish a foundation from which a general glycosyltransferase mechanistic consensus can be derived. In addition, the selected reactions (alpha/beta 1 yields 4 mannosyl transfer) are analogous to those found in eukaryotic N-linked glycoprotein biosynthesis and mechanism-based inhibitors based upon these models should lead to altered eukaryotic cell surface glycosylation patterns terminating in N-acetyl-D-glucosamine (GlcNAc) and Man, defining the cell as "non-self" and thus providing therapeutic opportunities. The beta-mannosyl linkage is also particularly difficult to prepare via chemical synthesis and our proposed studies may lead to enzymatic alternatives. Finally, the presented in vitro evolution/selection methodology should contribute to the general synthetic utility of glycosyltransferases (by providing simplified substrates and the potential to screen for essentially any glycosyl transfer event) and possibly lead to future bacterial cell surface engineering towards designed therapeutic vaccines.